Analysis of a novel multiplex polymerase chain reaction assay as a sensitive tool for the diagnosis of indeterminate and tuberculoid forms of leprosy.

OBJECTIVE/BACKGROUND: Clinical diagnosis of indeterminate and tuberculoid leprosy is often difficult due to limited and confounding signs and symptoms. In the current study, we evaluated the utility of new multiplex polymerase chain reaction (PCR) using Mycobacterium leprae-specific DNA sequences in the pseudogene regions of ML1545, ML2180, and ML2179 for PCR-based diagnosis of indeterminate leprosy (IND) and leprosy cases across the immunological spectrum. The sensitivity was compared with that of RLEP PCR. METHODS: DNA was extracted from paraffin-embedded skin biopsy specimens of 220 leprosy cases, which were divided into IND (41), tuberculoid form (3), borderline tuberculoid (42), midborderline (3), borderline lepromatous (n=59), and lepromatous leprosy (72) cases. PCR positivity of both multiplex and RLEP PCR were compared in all the samples. A decision tree was constructed using the classification and regression trees algorithm to predict the probability of PCR positivity with the new multiplex PCR scheme in various clinical groups of leprosy. Sensitivity of each pseudogene target was determined using real-time PCR assays, and specificity was confirmed by PCR amplification of DNA extracted from three other mycobacterial species and skin biopsies of 44 non-leprosy cases. RESULTS: A multiplex PCR positivity of 75.61% was noted in IND cases when compared to that of 58.54% using RLEP PCR (P < 0.05). Enhanced multiplex PCR positivity was noted across various clinical groups in comparison to RLEP PCR. The decision tree classifier has predicted statistically significant probability for multiplex PCR positivity among RLEP-PCR negative group and clinical groups with a low bacillary load. CONCLUSION: This new multiplex PCR scheme can support the diagnosis of indeterminate and tuberculoid forms of leprosy with limited clinical manifestations and can be implemented in basic clinical/diagnostic setting that possess conventional PCR facilities.

Genomic diversity in Mycobacterium leprae isolates from leprosy cases in South India.

OBJECTIVE: The Objective of this study was to identify the strain diversity of Mycobacterium leprae in terms of SNP types and subtypes stratified as per genomic single nucleotide polymorphisms, in clinical isolates of leprosy patients from a tertiary care leprosy center in South India. Further, the associations of SNP types with clinical outcomes in leprosy were also investigated. METHODS: DNA was extracted from excisional skin biopsies of a total of 172 newly diagnosed untreated leprosy patients from a clinic in Tamil Nadu, in south India, that also serves patients from neighboring states. All the leprosy patients were those who voluntarily reported at the clinic during the study period of one year i.e., 2015. Clinical and histopathological details were collected at diagnosis and leprosy was confirmed through bacteriological smear examination and PCR for M. leprae specific RLEP region. SNP types and subtypes were determined by PCR amplification and Sanger sequencing of PCR products. RESULTS: M. leprae specific RLEP gene amplification was achieved in 160 out of 172 patients. Among 160 specimens 118(73.75%) were type 1 and 42 (26.25%) were type 2 and on subtyping it was noted that 88/160 (55.00%) were 1D, 25/160 (15.62%) 1C, 5/160 (3.12%) 1A, 33/160 (20.62%) 2G and 9/160 (5.62%) were 2H. CONCLUSION: Our results indicated that subtype 1D is predominant in the south Indian population. We also noted 2G, 1C and 1A in the patient sample tested. Additionally we identified subtype 2H for the first time in India.

Computational Modelling of Dapsone Interaction With Dihydropteroate Synthase in Mycobacterium leprae; Insights Into Molecular Basis of Dapsone Resistance in Leprosy.

The molecular basis for determination of resistance to anti-leprosy drugs is the presence of point mutations within the genes of Mycobacterium leprae (M. leprae) that encode active drug targets. The downstream structural and functional implications of these point mutations on drug targets were scarcely studied. In this study, we utilized computational tools to develop native and mutant protein models for 5 point mutations at codon positions 53 and 55 in 6-hydroxymethyl-7, 8-dihydropteroate synthase (DHPS) of M. leprae, an active target for dapsone encoded by folp1 gene, that confer resistance to dapsone. Molecular docking was performed to identify variations in dapsone interaction with mutant DHPS in terms of hydrogen bonding, hydrophobic interactions, and energy changes. Schrodinger Suite 2014-3 was used to build homology models and in performing molecular docking. An increase in volume of the binding cavities of mutant structures was noted when compared to native form indicating a weakening in interaction (60.7 Å(3) in native vs. 233.6 Å(3) in Thr53Ala, 659.9 Å(3) in Thr53Ile, 400 Å(3) for Thr53Val, 385 Å(3) for Pro55Arg, and 210 Å(3) for Pro55Leu). This was also reflected by changes in hydrogen bonds and decrease in hydrophobic interactions in the mutant models. The total binding energy (ΔG) decreased significantly in mutant forms when compared to the native form (-51.92 Kcal/mol for native vs. -35.64, -35.24, -46.47, -47.69, and -41.36 Kcal/mol for mutations Thr53Ala, Thr53Ile, Thr53Val, Pro55Arg, and Pro55Leu, respectively. In brief, this analysis provided structural and mechanistic insights to the degree of dapsone resistance contributed by each of these DHPS mutants in leprosy.